Project Summary Craniofacial anomalies constitute a third of all congenital malformations. Orofacial clefting (OFC), one of the most common craniofacial birth defects, occurs in as many as 1 in 700 births. OFC is a multifactorial disease caused by numerous genetic and environmental factors, and there is a continued need to identify genes that contribute to its pathogenesis. Heterozygous mutations in SPECC1L, a novel cytoskeletal gene, have been identified in patients with atypical, syndromic, and non-syndromic OFCs, suggesting it may play an important role in craniofacial morphogenesis. Severe Specc1l gene-trap mouse mutants exhibit embryonic lethality with reduced PI3K-AKT signaling and cranial neural crest cell (CNCC) delamination failure. My data show that mouse embryos with moderate Specc1l deficiency exhibit perinatal defects, including cleft palate. CNCCs are a critical cell population for craniofacial development, including proper palate formation. I hypothesize that the palate closure defects seen upon moderate Specc1l deficiency are due to defects in CNCC function post-delamination, either in proliferation, survival, migration, or differentiation. In Specific Aim 1, I will test each aspect of CNCC function via two different methods. First, in vitro wound healing assays suggest Specc1l deficiency results in reduced collective cell migration. To test this in vivo, the delamination and collective migration of GFP-labeled CNCCs in Specc1l mutant embryos will be measured by live-embryo imaging and quantitative analysis. Second, a tissue-level analysis of the palatal region will be performed on moderate Specc1l mutant embryos to test for changes in proliferation, apoptosis, and differentiation. In addition, since severe mutants exhibit lower PI3K-AKT signaling, pan-AKT and phospho-AKT will also be measured. Next, Specific Aim 2 will test the hypothesis various Specc1l-deficient phenotypes can be rescued in vivo by upregulating PI3K-AKT signaling. First, PI3K-AKT signaling levels will be increased genetically in our Specc1l mouse mutants by crossing them with mice heterozygous for the tumor suppressor Phosphatase and tensin homolog (PTEN), a negative regulator of AKT phosphorylation. The rescue efficiency will be determined by gross assessment and histological marker analysis of embryos from the resultant matings. In addition, the collective migration of mouse embryonic palatal mesenchyme cells isolated from these embryos will be assessed by wound-repair analysis. The experiments in the second part of this aim will test the ability of small-molecule AKT-activator SC79 to rescue the collective CNCC delamination and migration defects seen in Specc1l mutants. Embryos will be treated with SC79 in utero by IP injections of the pregnant dam and cultured with SC79 during live-embryo imaging. The experiments in this proposal aim to provide insight into the role of SPECC1L in craniofacial development, expand the field?s understanding of OFC etiology, and explore the potential of PI3K-AKT signaling as a therapeutic target for OFC treatment.